Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation

The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca²⁺ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca²⁺ induced. These data underline the importance of PR70-Ca²⁺ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.     

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10⁻⁶, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5–10, p = 3.3 x 10⁻⁶, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.     

A β‐amino acid modified heptapeptide containing a designed recognition element disrupts fibrillization of the amyloid β‐peptide

We study the complex formation of a peptide βAβAKLVFF, previously developed by our group, with Aβ(1–42) in aqueous solution. Circular dichroism spectroscopy is used to probe the interactions between βAβAKLVFF and Aβ(1–42), and to study the secondary structure of the species in solution. Thioflavin T fluorescence spectroscopy shows that the population of fibers is higher in βAβAKLVFF/Aβ(1–42) mixtures compared to pure Aβ(1–42) solutions. TEM and cryo‐TEM demonstrate that co‐incubation of βAβAKLVFF with Aβ(1–42) causes the formation of extended dense networks of branched fibrils, very different from the straight fibrils observed for Aβ(1–42) alone. Neurotoxicity assays show that although βAβAKLVFF alters the fibrillization of Aβ(1–42), it does not decrease the neurotoxicity, which suggests that toxic oligomeric Aβ(1–42) species are still present in the βAβAKLVFF/Aβ(1–42) mixtures. Our results show that our designed peptide binds to Aβ(1–42) and changes the amyloid fibril morphology. This is shown to not necessarily translate into reduced toxicity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345     

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.     

Streptococcus pneumoniae Coinfection Is Correlated with the Severity of H1N1 Pandemic Influenza

Background

Initial reports in May 2009 of the novel influenza strain H1N1pdm estimated a case fatality rate (CFR) of 0.6%, similar to that of seasonal influenza. In July 2009, however, Argentina reported 3056 cases with 137 deaths, representing a CFR of 4.5%. Potential explanations for increased CFR included virus reassortment or genetic drift, or infection of a more vulnerable population. Virus genomic sequencing of 26 Argentinian samples representing both severe and mild disease indicated no evidence of reassortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence. Furthermore, no evidence was found for increased frequency of risk factors for H1N1pdm disease.

Methods/Principal Findings

We examined nasopharyngeal swab samples (NPS) from 199 cases of H1N1pdm infection from Argentina with MassTag PCR, testing for 33 additional microbial agents. The study population consisted of 199 H1N1pdm-infected subjects sampled between 23 June and 4 July 2009. Thirty-nine had severe disease defined as death (n = 20) or hospitalization (n = 19); 160 had mild disease. At least one additional agent of potential pathogenic importance was identified in 152 samples (76%), including Streptococcus pneumoniae (n = 62); Haemophilus influenzae (n = 104); human respiratory syncytial virus A (n = 11) and B (n = 1); human rhinovirus A (n = 1) and B (n = 4); human coronaviruses 229E (n = 1) and OC43 (n = 2); Klebsiella pneumoniae (n = 2); Acinetobacter baumannii (n = 2); Serratia marcescens (n = 1); and Staphylococcus aureus (n = 35) and methicillin-resistant S. aureus (MRSA, n = 6). The presence of S. pneumoniae was strongly correlated with severe disease. S. pneumoniae was present in 56.4% of severe cases versus 25% of mild cases; more than one-third of H1N1pdm NPS with S. pneumoniae were from subjects with severe disease (22 of 62 S. pneumoniae-positive NPS, p = 0.0004). In subjects 6 to 55 years of age, the adjusted odds ratio (OR) of severe disease in the presence of S. pneumoniae was 125.5 (95% confidence interval [CI], 16.95, 928.72; p<0.0001).

Conclusions/Significance

The association of S. pneumoniae with morbidity and mortality is established in the current and previous influenza pandemics. However, this study is the first to demonstrate the prognostic significance of non-invasive antemortem diagnosis of S. pneumoniae infection and may provide insights into clinical management.     

Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium

In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.